Hepatitis C virus (HCV) is a member of the hepacivirus genus in the Flaviviridae family and has infected 180 million people worldwide amongst whom 130 millions are chronic carriers at risk of developing liver cirrhosis or hepatocellular carcinoma. With an incidence of 3-4 million infections per year, HCV is responsible for 50-76% of all liver cancer cases and two thirds of all liver transplants in the developed world. The current standard of care, consisting of pegylated interferon-α2 combined with ribavirin, results in about 50% sustained virological response (Moradpour, D. et al., 2007. Nat. Rev. Microbiol. 5, 453-463). It is even less efficient against subtypes 1b and 1a, the two most prevalent in Europe and North America, respectively. (Manns, M. P. et al., 2007. Nat. Rev. Drug Discov. 6, 991-1000).
The virus replicates at a very rapid rate (1012 virion/day) (Layden, T. J. et al., 2000. Semin. Liver Dis. 20, 173-183) and viral polymerase's lack of proof reading activity results in the emergence of viral quasispecies rapidly evolving with any kind of selective pressure. Several viral protease and polymerase inhibitors are now in the latter stages of clinical development and, despite their potent antiviral effects, have been complicated by the rapid selection of drug-resistance mutants (Thompson & McHutchison. 2009. J. Viral Hep. 2009. 16, 377-387). For similar reasons, there is no effective vaccine available yet, in vivo. Not surprisingly, antibody against hypervariable region 1 (HVR 1) of E2 failed to protect against the emergence of neutralization escape mutants (Farci, P. et al., 1996. Proc. Natl. Acad. Sci. USA 93, 15394-15399). In addition, serum antibodies from chronically HCV-infected individuals demonstrate broadly neutralizing properties in vitro and yet fail to control infection in vivo (Timpe, J. et al., 2008. Hepatology 47, 17-24). Anti-Envs antibodies have even been reported to enhance in vitro infectivity (Meyer, K. et al., 2008. J. Virol. 82, 2140-2149), as previously described for flaviviruses (e.g. West Nile (WN) and dengue viruses), or fail to inhibit cell-to-cell transmission (Timpe, J. M. et al., 2008. Hepatology 47, 17-24). Alternative antiviral strategies less prone to quickly select viral mutants are in high need; other steps of the HCV life cycle, such as cellular factors involved in viral production (assembly/release) and entry/genome uncoating, could represent such alternative.
HCV genomic RNA directly encodes an 3,000-amino-acid polyprotein that is processed by cellular as well as viral proteases. The first part of the genome encodes the structural proteins: core likely forms the nucleocapsid in viral particles (Yasui, K. et al., 1998. J. Virol. 72, 6048-6055) and E1, E2 envelope glycoproteins carry the fusogenic activities required for viral entry (Penin, F. et al., 2004. Hepatology 39, 5-19; Voisset, C., et al., 2004. Biol. Cell. 96, 413-420). p7 and NS2 are required for viral assembly and/or egress (Steinmann, E., F. et al., 2007. PLoS Pathog 3, e103; Jones, C. T. et al., 2007. J. Virol. 81, 8374-8383). The non-structural proteins NS3 to NS5B, together with 5′- and 3′-untranslated regions, support the viral replication (Lohmann, V. et al., 1999. Science 285, 110-113; Blight, K. J. et al., 2000. Science 290, 1972-1974). Soluble, truncated HCV E2 was used to identify cell surface binding molecules such as CD81 (Pileri, P. et al., 1998. Science 282, 938-941) and SR-BI (Scarselli, E. et al., 2002. Embo J. 21, 5017-5025) involved in HCV entry. Pseudo-typing retrovirus with HCV envelope proteins, Bartosch et al (2003. J. Exp. Med. 197, 633-642) demonstrated that released particles (HCVpp) could enter target cells via CD81, SR-BI and claudin-1 (Evans, M. J. et al., 2007. Nature 446, 801-805) and occludin (Ploss, A. et al., 2009. Nature 457, 882-886)-dependent mechanisms. Lastly, a strain of genotype 2a (JFH-1; Kato, T. et al., 2001. J. Med. Virol. 64, 334-339) was identified reproducing a full infectious cycle in replication-permissive hepatocellular carcinomas cells in culture (Wakita, T. et al., 2005. Nat. Med. 11, 791-796; Zhong, J. et al., 2006. J. Virol. 80, 110892-11093).
There remains a need for a system for production of HCV particles that may be employed with multiple genotypes. There also remains a need for a method of uncoupling viral entry from its replication in order to specifically study the interactions of viral envelopes with target cell surface molecules and their involvement in internalization mechanisms. This method theoretically will also allow studying HCV production steps (translation, assembly and egress) and their mechanisms without interference from viral RNA replication. In essence, this method could lead to the identification of cellular factors that are specifically involved in HCV assembly and egress.